Methods of collecting cells from multi-well plates for use in flow cytometry

ABSTRACT

A method of collecting cells from individual wells of a multi-well plate for flow cytometry, the method comprising: determining particle or cell counts from each well of the multi-well plate by flow cytometry; ordering the wells from lowest particle count to highest particle count; generating a sequential order for collection that follows the ordering of wells to establish a collection pattern; and collecting particles or cells according to the collection pattern in a second multi-well plate by flow cytometry.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/750,613 filed Jun. 25, 2015, which claims the benefit of priorityunder 35 U.S.C. § 119(e) of U.S. provisional patent application Ser. No.62/017,251, filed Jun. 25, 2014; the content of each is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to the field of flow cytometry and morespecifically to methods of sample collection from multi-well plates foruse with flow cytometry systems.

BACKGROUND OF THE INVENTION

Flow cytometry is a laser-based, biophysical technology wherefluorescent molecules coupled to cells are aspirated into the flowcytometer, then passed one by one through a flow cell and excited by aset of lasers. The emitted fluorescence is then collected andtransformed into an electrical signal for analysis. Labelling cells withmolecules that fluoresce at different wavelengths allows the user toidentify a variety of distinct cell populations and therefore itprovides a powerful tool with diagnostic, therapeutic, and researchapplications. For example, the technique is often used to count cellsfrom a biological sample, such as counting CD4/CD8 populations fromcirculating blood in human immunodeficiency virus (HIV) studies to mapthe disease, and to sort cells from a mixed population of cells, such asstem cell sorting from harvested biological samples for possibledifferentiation and reintroduction into different areas of the patient'sbody.

Since numerous distinct cell populations can be identified using flowcytometry often a single flow cytometry experiment or test includes alarge number of samples. Therefore, there is a need to increase thethroughput of flow cytometers. One solution is to present test sampleson multi-well plates and program a robot to acquire samples across themulti-well plates, which is conventionally performed by aspirating cellsamples from the wells. However, a challenge with presenting cells inmultiwall plates is that cells tend to settle from solution and collectat the bottom of wells while waiting for aspiration, thereby decreasingcell yields in later aspirated samples.

A possible solution to the problem of cell settling is to rotate oragitate multi-well plates at various intervals to assist in suspendingcells for aspiration. We attempted different intervals including betweensteps of aspirating samples from different wells. However, it has nowbeen found that merely using a conventional suspension approach ofrotating or agitating the multi-well plate, even at frequent intervals,only significantly helps suspend cells positioned at opposing ends ofthe plate. Importantly, it is found to be insufficient to suspend cellspositioned within the middle region of the plates. That is, the approachtends to suspend cells in the outer region of the multi-well plate butfails to adequately suspend cells at the middle region of the plate.Accordingly, there is a need to develop an approach useful in highthroughput sample acquisition for flow cytometry procedures that canincrease the consistency of cell yields across the entire multiwallplate.

BRIEF SUMMARY OF THE INVENTION

The above deficiencies in sample acquisition are addressed by themethods of the invention. More specifically, the methods accomplish atleast two objectives related to high throughput sampling of multi-wellplates in flow cytometry applications. The first is to improveconsistency of particle or cell counts across the entirety of the plate.The second is to alleviate the issue of inconsistent mixing across theplate by agitation or plate shaking.

The above is achieved through a method of collecting cells fromindividual wells of a multi-well plate for use in a flow cytometryapplications, which includes adding a suspension of cells to the wellsof the multi-well plate; and aspirating cells from different wellsaccording to a collection pattern into a flow cytometer, where thecollection pattern is a sequential ordering of wells beginning at amiddle region of the multi-well plate and continuing towards an outerregion of the multi-well plate. In preferred embodiments the methodfurther includes rotating or agitating the multi-well plate betweensteps of aspirating cells from different wells.

The method is useful for any cell or particle suitable for flowcytometry systems. Typically cells or particles will be sized between0.2 μm to 150 μm and the methods will become still more useful as cellstend to settle from solution at higher rates. The method can be usedwith prokaryotic cells but is preferably used with eukaryotic cells andin particular human cells. To this end, the method can be used whendetecting, counting or sorting any cell or particle populationconsistent with flow cytometry systems including cancer cells,monocytes, lymphocytes, and other cells isolated from tissue or grown incultures sized between 0.2 μm to 150 μm. As known in the flow cytometryarts, the cells may be labelled with one or more labelled bindingreagents against one or more cell biomarkers or exposed to one or moredetectable intercalating agents. In some embodiments, the labelledbinding reagents are fluorescently labelled antibodies or antibodyfragments and the intercalating agent is propidium idodide. In othermethods the cells are not labelled, such as experiments where onlyforward scatter (FSC) and side scatter (SSC) data is required.

In preferred embodiments, the multi-well plate is a 96 well plate,characterized as having rows A-H and columns 1-12. Preferably, themiddle region is characterized as a well selected from the groupconsisting of wells D6, D7, E6 and E7. In some embodiments, cells fromwells B4-B8, C4-C8, D4-D8, and E4-E8 are aspirated before cells fromremaining all wells in a same 96 well plate. Still further, in someembodiments, cells from wells C5-C7, D5-D7, and E5-E7 are aspiratedbefore cells from all remaining wells of a same 96 well plate. Inembodiments where two or more plates are provided, the methods mayaspirate samples from the above wells from different plates prior toaspirating cells from all remaining wells of the different plates or mayaspirate all wells from a same plate prior to aspirating cells from adifferent plate.

In some embodiments, the collection pattern is a spiral-squareconfiguration, which is characterized as a series of wells in successivesquare patterns that each circle the middle region and where thesequential ordering proceeds by aspirating adjacent wells. When themulti-well plate is a 96 well plate, the spiral-square configuration caninclude three successive square patterns around a central well, such aswell selected from the group consisting of wells D6, D7, E6 and E7 butpreferably D6. In some embodiments, the spiral-square configuration isfurther characterized as successively aspirating cells from wells thatare immediately adjacent to the prior well that was aspirated for atleast one half of all wells in the 96 well plate.

In an exemplary embodiment, the multi-well plate is a 96 well platedefined by rows A-H and columns 1-12, and the sequential orderingincludes the well order D6, E6, E7, D7, C7, C6, C5, D5, E5, F5, F6, F7,F8, E8, D8, C8, B8, B7, B6, B5, B4, C4, D4, E4, F4, G4, G5, G6, G7, G8,G9, F9, E9, D9, C9, B9, A9, A8, A7, A6, A5, A4, A3, B3, C3, D3, E3, F3,G3, H3, H4, H5, H6, H7, H8, H9, H10, G10, F10, E10, D10, C10, B10, A10.In one variation, the order further includes the order of A2, A1, B1,B2, C2, C1, D1, D2, E2, E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11,F11, F12, E12, E11, D11, D12, C12, C11, B11, B12, A12, A11. In anothervariation, the order further includes meandering or alternating betweenrows of column 1 and column 2 or rows of column 11 and column 12.

In other embodiments the collection pattern is a nearest well to centerpattern, which is characterized as successively aspirating cells from awell that is nearest to the middle region, where the middle region isdefined as a center of the multi-well plate independent of whether awell is positioned at the center. A modified nearest well to center is avariation on the embodiment and is characterized as successivelyaspirating cells from a well that is nearest to both the middle regionand a well from which an immediately prior cell was aspirated.

In another exemplary embodiment the multi-well plate is a 96 well plateand the collection order is D6, E6, E7, D7, C7, C6, D5, E5, F6, F7, E8,D8, C8, C5, F5, F8, E9, D9, B7, B6, D4, E4, G6, G7, G8, F9, C9, B8, B5,C4, F4, G5, H6, H7, E10, D10, A7, A6, D3, E3, G4, G9, B9, B4, C3, F3,H5, H8, F10, C10, A8, A5, A4, B3, G3, H4, H9, G10, B10, A9, D2, E2, E11,D11, C11, F11, F2, C2, A3, H3, H10, A10, B11, G11, G2, B2, D1, E1, E12,D12, C12, F12, F1, C1, A2, H2, H11, A11, B12, G12, G1, B1, A1, H1, H12,and A12.

The collection pattern may include two or more distinct patterns. Insome embodiments the first pattern is a square pattern or spiral-squarepattern and a second pattern is a meandering pattern.

The collection patterns themselves may be programmed by the user into asample collection or acquisition software module or can be preloaded inflow cytometer as one or more presets, which provides the user with aselectable option to use one or more sample collection patterns.Alternatively, a sample collection module of sample acquisition softwaremay automatically perform the collection patterns. The software may alsoassign the rotation or shaking interval of the multi-well plate.

In a related embodiment, the invention provides a method of collectingcells from individual wells of a multi-well plate for flow cytometry,the method comprising determining particle or cell counts from each wellof the multi-well plate by flow cytometry; ordering the wells fromlowest particle count to highest particle count; generating a sequentialorder for collection that follows the ordering of wells to establish acollection pattern; and collecting particles or cells according to thecollection pattern in a second multi-well plate by flow cytometry. Inpreferred embodiments, the method also includes rotating or agitatingthe multi-well plate between steps of collecting particles or cells fromdifferent wells. In some embodiments the pattern exactly follows theordering of wells from lowest particle count to highest particle count;however, in other embodiments the collection pattern deviates from theordering of wells less than 15%.

BRIEF DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the invention but instead to demonstrate theimprovements over the state of the art and various exemplaryembodiments.

FIG. 1 is table representing a 96 well plate, which shows a collectionpattern including both a spiral-square pattern and a followingmeandering pattern. An exemplary sequential ordering of each well isdemonstrated.

FIG. 2 is table representing a 96 well plate, which shows a collectionpattern including a nearest well to center pattern. An exemplarysequential ordering of each well is demonstrated.

FIG. 3 is a series of flow cytometry plots comparing forward scatter andside scatter for polystyrene beads and gated populations E1 and E2 forwell F7 using the collection pattern of FIG. 1.

FIG. 4 is a table displaying the measured counts of silica particlesfrom each well of a 96 well plate using a row-by-row collection pattern(proceeding from row A to row H) and corresponding statistics.

FIG. 5 is a table displaying the measured counts of silica beads fromeach well of a 96 well plate using a spiral-square collection patternand corresponding statistics.

FIG. 6 is a table displaying the measured counts of silica beads using anearest to center collection pattern, in which the wells are sampled inorder based on the nearest distance from the center of the 96 wellplate.

FIG. 7 is a table displaying the measured counts per well of polystyrenebeads using a spiral-square collection pattern and correspondingstatistics.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, the preferred methods andmaterials are now described. To facilitate understanding of theinvention, a number of terms and abbreviations as used herein aredefined below as follows.

The term “multi-well plate” as used herein refers to a plate having aplurality of wells capable of retaining a population of cells having asuitable working volume for flow cytometry sample aspiration. Multi-wellplates are commonly available in formats of 6 well, 12 well, 24 well, 48well, 96 well, 384 well and 1536 well and can be flat bottom or roundedbottom. Conventional 6 and 12 well plates have working volumes of about1.9-2.9 mL and 0.76-1.14 mL respectively and are not typically used forhighthroughput flow cytometry. Conventional 48 well plates have aworking volume of about 190-285 μL and therefore may be used but are nottypically preferred due to the desire to increase the number ofavailable wells thereby increasing throughput. Conventional 96 wellplates have a working volume of about 100-200 μL and are preferred asthey balance the needs of high throughput with sufficient sample volumefor high throughput flow cytometry. Conventional 384 well plates havinga working volume of 25-50 μL and may be suitable but much less preferredas the sample volumes are on the low end of flow cytometry applications.1536 well plates having a working volume of 5-10 μL and are thereforemuch less desirable. Most preferably, the multi-well plate is a 96 wellplate having a rounded bottom.

The term “row-by-row” as used herein refers to a collection pattern thatproceeds across a single row, then proceeds across a neighboring row,and so on until all rows are aspirated. In the case of a conventional 96well plate, cells or particles from wells assigned to column 1-12 of therow “A” are typically aspirated first, followed by the aspiration ofcells or particles from wells assigned to columns 1-12 of row “B”, thenrow “C”, and so on until aspirating samples from all columns across eachrow.

The term “column-by-column” as used herein refers to a collectionpattern that proceeds up or down a single column, then proceeds up ordown the neighboring column and so on. In the case of a 96 well plate,samples from each row of the column conventionally referred to as column“1” are typically aspirated first, followed by the aspiration of cellsor particles from each row of column “2”, then column “3”, and so onuntil cells are aspirated from all rows of each column.

The term “spiral-square” as used herein is a collection pattern thatfollows a sequence of successive square patterns in a spiral order. Thespiral order can be a right handed spiral or a left handed spiral, whichspirals to the right or left.

The term “sequential ordering” as used herein refers to the order ofwells for aspiration.

The term “middle region” as used herein refers to the center most pointof the multi-well plate together with immediately adjacent wells.Preferably, the middle region is characterized as having four (4) orfewer wells. If there are an even number of rows or columns, such as ona 96 well plate, the middle region can be the combination of wells thatform the center of both the rows and columns (e.g. a region defined byD6, D7, E6, and E7 on a 96 well plate), any two of the combination ofwells (e.g. a region defined by one of D6 and D7, E6 and E7, D6 and E6,D6 and E7, E6 and E7) or any single well of the combination (e.g. D7,D7, E6, or E7).

The term “continuing towards” as used herein refers to a progressionover a series of wells.

The term “successive pattern” as used herein refers a pattern of wellsthat maintains a prior pattern of wells but differs in size. “Successivesquare pattern” in a 96 well plate can include a first square patternformed from wells E5-E7, C7-E7, C5-C7 and C5-E5; followed by a secondsquare pattern formed from wells F4-F8, B8-F8, B4-B8, and B4-F4; andfollowed by a third square pattern formed from wells G3-G9, A9-G9, A3-A9and A3-G3.

The term “immediately adjacent” as used herein refers to the positioningof two wells next to one another without an intervening well.

The term “well that is nearest to the center of the multi-well plate andfrom which an immediately prior cell was aspirated” as used herein tothe well that has not yet been aspirated and that compared to remainingwells that have not yet been aspirated has the lowest sum of distancesto the both the center of the multi-well plate and to the well fromwhich a sample was last aspirated.

The term “selectable preset” as used herein refers to a software optionthat encodes the collection pattern that may be selected by a userwithout additional programming.

The term “biomarker” as used herein refers to a detectable moiety of acell. A biomarker may be a cluster of differentiation (CD) for a cell,such as a glycoprotein found on a surface of a cell, can be a nucleicacid, or any other moiety useful for detecting one or more cells in flowcytometry.

As an introduction to the invention, among the challenges presentedduring sample acquisition in high throughput flow cytometry systems thatuse multi-well plates is maintaining consistent particle or cell countsacross all wells of the plate. In particular, as cells wait foraspiration they tend to settle to the bottom of the well. While one ofordinary skill in the art may consider rotating or agitating the plateusing a rotating platform or shaking platform, Example I demonstratesthis does not adequately suspend cells across the entirety of themulti-well plate. More specifically, Example I and corresponding FIG. 4demonstrate the need to address inconsistencies in particle count orcell count across different wells of a 96 well plate even when rotatingor agitating the multi-well plate.

In Example I, cells were added in equal amounts to each well across themulti-well plate. Wells were aspirated, and the plate was rotated oragitated between aspirating each well. The collection pattern used was aconventional a row-by-row sample collection method, where samples werecollected across row A before proceeding to row B and so on. There was asignificant discrepancy found in particle counts across the wells. Thatis, particle counts were found to be inconsistent across the plate whenusing a row-by-row approach even with rotating or agitating the plateafter aspirating each well. In particular, as shown in FIG. 4, datacollected from wells at about the middle columns of the plate hadsignificantly fewer counts than wells located at end columns. Forinstance, the average particle counts from columns 1-3 and 10-12 weresignificantly higher than the average particle counts for columns 4-6and 7-9. Further, the average particle counts at the middle mostcolumns, namely columns 5-7 were the lowest overall. Still further,aspirates from wells D7, E4, E6, E7, F5-7, and G4-8 did not yield anyparticle counts. Since middle regions of the plate are shown to benefitthe least from rotation or shaking of the multi-well plate, the objectof the invention is to devise an approach to increase particle or cellcounts at the middle region of the multi-well plate. As to 96 wellplates, the primary challenge is to increase counts at wells closest towells D6, D7, E6 and E7, where plate rotation and agitation alone wasfound to be the least effective. For completeness, although not shown,column-by column sample collection also suffers from a same problem.

As shown in FIGS. 5-7, significant improvement to increasing particlecounts across the entire multi-well plate has been achieved through anew approach of sample collection for flow cytometry. In particular, amethod of collecting cells or particles from individual wells of amulti-well plate for use in flow cytometry system has been achieved. Themethod includes adding a suspension of cells to wells of the plate; andaspirating cells from different wells according to a collection patterninto a flow cytometer, wherein the collection pattern is a sequentialordering of wells beginning at a middle region of the multi-well plateand continuing towards an outer region of the multi-well plate. Byaltering the collection pattern to collect samples from the middleregion of the plate first, particles at greatest risk of settling fromsolution can be collected prior to the settling occurring. Combiningthis collection pattern approach with rotating or shaking the multi-wellplate to maintain the suspension of cells in outer wells surprisinglyincreased the consistency of particle counts across the entire plate. Tothis end, in preferred embodiments the method further includes rotatingor agitating the multi-well plate between steps of aspirating cells fromdifferent wells, a least those along the outermost regions of the plate.The rotating or agitating can be performed using a rotating platform orshaking platform incorporated into a multi-well plate sampling chamberof a flow cytometer and the rotation or shaking feature can beprogrammed to occur between aspirating different wells. Further, therotating or shaking platform can also be programmed to position therequired well under an aspiration needle of a flow cytometer.

Although the examples demonstrate the improvements over the art withrespect to particles and in particular to silica beads or polystyrenebeads, the method is equally useful for cell analysis. The similarity oftested 5 μm polystyrene beads (Spheroteach Accucount Beads) to cells isdepicted in FIG. 3, where the forward scatter (FSC) and side scatter(FSC) of the particles is depicted. The gated E1 and E2 populationsmatch the parameters for human cells. Like particles, cells tend tosettle from solution and collect at the bottom of multi-well plates.Further, it has also been observed that agitating or rotating multi-wellplates is significantly more effective at suspending cells that are atend columns compared to middle columns and at end rows compared tomiddle rows when the plate is positioned at the middle of the rotatingor shaking apparatus. As further guidance for the skilled artisan, themethod will be useful when aspirating cells or particles sized between0.2 μm to 150 μm. The skilled artisan will further appreciate that themethod is useful for aspirating prokaryotic or eukaryotic cells and willhave particular use for aspirating human cells, whether normal, infectedwith disease, cancerous cells or at any stage of development.

The skilled artisan will also appreciate that cells can be harvest fromany suitable fluid or organ as known in the flow cytometry arts, such asblood, serum or tissue biopsy. Cells can be harvested from adiposetissue. Alternatively, the cells can be from cell lines as known in theart to which the invention belongs. To this end, the method can be usedwhen detecting, counting or sorting any cell or particle populationconsistent with flow cytometry systems including cancer cells,monocytes, lymphocytes, and any other cells isolated from tissue orgrown in cultures sized between 0.2 μm to 150 μm and suited for flowcytometry applications.

Although the methods herein significantly improve counts across theentire multi-well plate and significantly improve counts from the middleregion of the multi-well plate, the methods may be optimized to achieveeven greater consistency in particle or cell accounts. Among thesemodifications the user may wish to optimize the number of cells added toeach well. That is, the artisan may find it beneficial to add fewer ormore cells across the multi-well plate, although it is generallypreferred to have the same number of cells in each well. Anothermodification may be to alter the volume in the well such as increasingthe sample volume or decreasing the sample volume, which may depend onthe concentration of cells in the well.

In some instances, cells will be added to the wells, aspirated accordingto the collection pattern and analyzed according to forward scatter(FSC) or side scatter (SSC) parameters. Such comparisons can be used toisolate monocyte populations from red blood cells. However, the skilledartisan will also appreciate that since flow cytometry often involveslabelling cells with one or more binding reagents for fluorescentdetection, the method will be especially useful in such procedures.Accordingly, prior to aspiration the cells may be labelled with one ormore labelled binding reagents against one or more cell biomarkers orexposed to one or more detectable intercalating agents. Consistent withthe flow cytometry arts, the labelled binding reagents can befluorescently labelled antibodies or fluorescently labelled antibodyfragments. There are many fluorescent labels from which the artisan maychoose, of which flourescein (FITC), phycoerythrin (PE) andallophycocyanin (APC) tend to be very popular. However, research anddevelopment groups are continually developing new fluorochromes for theflow cytometry arts and thus the availability of corresponding labelledbinding reagents are increasingly available through a variety ofcompanies, including but not limited to Becton Dickenson (FranklinLakes, N.J.), Jackson ImmunoResearch Laboratories, Inc (West Grove, Pa.)and many others known those skilled in the flow cytometry arts. Each ofthese can be used with the present invention. In some embodiments theintercalating agent is propidium idodide, which can intercalate into DNAmolecules. Again, such molecules are also commonly known in the art towhich the invention belongs and are included in the present invention.

In flow cytometry, cells are aspirated using an aspirating needle, whichdirects cells into the flow cytometer, where cells mix with sheath fluidand are streamed through a flow cell for excitation. As such, theinvention is directed towards maximizing the availability of cellsthrough the flow cell by provide new sample collection patterns thatincrease the consistency of cell counts or particle accounts across themulti-well plate. The term “collection pattern” as used herein refers tothe sequence or order for aspirating wells across the multi-well platefor collection by the flow cytometer and thus streaming through the flowcell.

In some embodiments, the collection pattern may be ordered beginningwith wells having a tendency to produce a lowest particle or cell countto wells having a tendency to produce a highest particle or cell count.This order can be determined experimentally by counting cells orparticles across the multi-well plate over a series of experiments whileagitating or rotating the plate. The wells can then be statisticallyordered from lowest particle or cell count to highest particle or cellcount and the order designated as a collection pattern for future samplecollection.

Alternatively, the collection pattern can include groups of wells thatare grouped according to count ranges then ordered such that groups withlower count ranges are aspirated before groups with higher count ranges.For example, a group of wells having a count range of 0-250 counts wouldprecede a group of wells with a count range of 250-500 counts, whichwould precede a group of wells with a count range of 500-1000 counts.This approach may be preferred when count variation between wells withina same count range continually alter the aspiration sequence using anabsolute lowest to highest ordering. That is, wells in a same region,such as the middle most region, may equally suffer from low cell orparticle counts and therefore their relative ordering with each othermay be less important compared to their ordering compared to wells ofdifferent regions on the multi-well plate.

In some embodiments, the collection pattern is a series of successivesquare patterns, where a first square pattern of wells is encircled by asecond square pattern of wells, and so on. In such a pattern the orderof aspirating wells in each square pattern may be randomly assigned ormay be further ordered such as ordered clockwise or counterclockwise.

In some embodiments, the collection pattern is a spiral-square pattern,which is characterized as a series of successive square patterns thatcircle the middle region or a central well with a caveat that moving toa next well in the collection sequence is performed by aspirating a wellthat is adjacent (either horizontally adjacent, vertically adjacent, ordiagonally adjacent) to the most recently aspirated well. An example isshown in FIG. 1, where a first square pattern is defined by thecollection sequence 2-9 and a second square pattern is defined by thecollection sequence 10-25, where moving outward from the first squarepattern to the second square pattern occurs at adjacent wells E5 and F5,which are ordered as collection sequence numbers 9 and 10. The skilledartisan will appreciate that the outward movement could alternatively beperformed between wells E5 and either wells D4, E4 or F4. Similarly,adjacent wells F4 and G4 in FIG. 1 are suited for the outward movementfrom the second square pattern (designated by order 10-25) to asuccessive third square pattern (designated by order 26-49); howeveroutward movement could also have been performed between wells F4 and E3,F3 or G3. Also as shown in FIG. 1, when the multi-well plate is a 96well plate, the spiral-square configuration can include three successivesquare patterns (e.g. defined by collection sequences 2-9, then 10-25,then 26-49) around a central well, in this case well D6. Whendetermining a middle region to begin a collection pattern for a 96 wellplate, the middle region or central well is preferably assigned to D6;however, any well between C5-C7, D5-D7 or E5-E7 of a 96 well plate maybe assigned to the middle region or center well of a 96 well plate andthus designated as a first well in the collection pattern. The skilledartisan will appreciate these modifications would shift the row andcolumn combination for the collection pattern but a same spiral-squarepattern could still be followed. The artisan will also appreciate thatthe spiral-square pattern can spiral to the left or right.

The artisan will also appreciate that the above collection pattern canalso be applied to multi-well plates having more or less than 96 wells.In such cases, the middle-most well can be chosen or chosen from a groupof middle most wells, and the collection pattern followed.

In some instances a square collection pattern or spiral-squarecollection pattern cannot be followed throughout the entire multi-wellplate because of the plate layout. That is, in some instances, such asthat of FIG. 1, the multi-well plate is not dimensioned such that asuccessive square or square-spiral pattern can collect samples from allwells. In such instances it is advisable to incorporate at least asecond pattern for aspirating outermost wells that cannot be collectedusing the first collection pattern. Such second patterns can be arectangular pattern that does not encircle the center well (e.g. apattern of collecting samples from column 2 followed by column 1 thencolumn 11 followed by column 12 in a 96 well plate) or as shown in FIG.1, a meandering pattern that moves sequentially between two neighboringcolumns along each row.

In an exemplary embodiment, the multi-well plate is a 96 well plate andthe collection order follows the wells D6, E6, E7, D7, C7, C6, C5, D5,E5, F5, F6, F7, F8, E8, D8, C8, B8, B7, B6, B5, B4, C4, D4, E4, F4, G4,G5, G6, G7, G8, G9, F9, E9, D9, C9, B9, A9, A8, A7, A6, A5, A4, A3, B3,C3, D3, E3, F3, G3, H3, H4, H5, H6, H7, H8, H9, H10, G10, F10, E10, D10,C10, B10, A10. In one variation, the order further comprises A2, A1, B1,B2, C2, C1, D1, D2, E2, E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11,F11, F12, E12, E11, D11, D12, C12, C11, B11, B12, A12, A11. In anothervariation, the order further comprises a second collection sequencealternating or meandering between column 1 and column 2 or column 11 andcolumn 12.

Another embodiment is depicted in FIG. 2, where the collection patternis a nearest to center pattern, where wells are ordered for aspirationby nearest proximity to a center of the multi-well plate independent ofwhether a well is positioned at the center of the multi-well plate. Asshown in FIG. 2, the center of the multi-well plate is positioned at theintersection of wells D6, D7, E6 and E7. The collection pattern thenbegins from a nearest well to a farthest well, and where the order ofwells that share a same distance (i.e. D6, D7, E6 and E7) can beinterchanged. In another variation if wells are equidistant from thecenter of the multi-well plate the pattern may further include a caveatthat the closest well to the well aspirated immediately prior to thequery follows next.

An exemplary embodiment of a nearest to center collection pattern isshown in FIG. 2, where in a 96 well plate and the well aspiration orderis D6, E6, E7, D7, C7, C6, D5, E5, F6, F7, E8, D8, C8, C5, F5, F8, E9,D9, B7, B6, D4, E4, G6, G7, G8, F9, C9, B8, B5, C4, F4, G5, H6, H7, E10,D10, A7, A6, D3, E3, G4, G9, B9, B4, C3, F3, H5, H8, F10, C10, A8, A5,A4, B3, G3, H4, H9, G10, B10, A9, D2, E2, E11, D11, C11, F11, F2, C2,A3, H3, H10, A10, B11, G11, G2, B2, D1, E1, E12, D12, C12, F12, F1, C1,A2, H2, H11, A11, B12, G12, G1, B1, A1, H1, H12, and A12.

As with prior embodiments, the nearest to center collection pattern canbe combined with a second pattern that differs from the first. In suchembodiments, preferably the nearest to center collection pattern isfollowed over the first 25% of wells, more preferably over first 50% ofwells and still more preferably of the first 75% of wells.

Similarly, the symmetry of a multi-well plate may permit the collectionpattern to proceed to the left or to the right, each of which isencompassed herein.

In other embodiments a collection pattern deviates less than 15% fromone of the collection patterns disclosed, where the deviation from acollection pattern is determined by comparing the collection sequencesand calculating the percent difference. If the percent difference iswithin 15 percent it is within the corresponding collection pattern.

Since collection patterns themselves are sets of ordered sequences, thecollection patterns may be programmed by the user into a samplecollection software module for use with a flow cytometer or can beprovide as one or more collection presets in the flow cytometer, whichprovides the user with a selectable option to use one or more collectionpatterns. Alternatively, the sample collection module of sampleacquisition software may automatically perform the collection patternsherein.

Collection patterns have been followed by adapting a currently availablemulti-sampling flow cytometry system, namely, the NOVOCYTE flowcytometer offered by ACEA BIOSCIENCES, INC. together with a samplecollection software program.

Depending on the adjustability of the user's flow cytometer system, theartisan may follow the disclosed patterns of sample collection toinitiate suitable collection pattern programming. In other embodiments,software designers may preprogram software to collect samples accordingthe patterns disclosed herein and optionally provide the user with oneor more selectable options or patterns for sample collection. Theskilled artisan will appreciate that the programming of collectionpatterns herein is not limited to any particular program language asprogramming could differ between flow cytometry systems. In someembodiments, the programming may adapt a Cartesian coordinate systemwhere plots along perpendicularly aligned axes correspond to welllocations.

EXAMPLES

Embodiments of the invention may be further understood in light of thefollowing examples, which are not to be construed as limiting the scopeof the disclosure in any way.

Example I Significant Variability in Particle Count was Found UsingRow-by-Row Collection Pattern for Flow Cytometry

The inventions addresses inconsistencies in cell counts found tocontinually occur when using a row by row or column by column basedmethod in sample acquisition. The following example demonstrates theinconsistencies found in wells across a 96 well plate when measuringcell counts using a row by row sample acquisition approach using theNOVOCYTE flow cytometer (ACEA Biosciences, Inc., San Diego, Calif.).

A suspension of silica beads was vortexed to ensure the beads wereequally distributed throughout the solution. An equal volume of thesuspension was aliquoted into each well of a 96 well plate to ensure anequal load across all wells. The NOVOCYTE flow cytometer (ACEABIOSCIENCES, San Diego, Calif.) was programmed to sequentially acquire10 uL of sample from each well and count the silica beads in eachacquired sample. Between acquiring samples from each well, the plate wassubjected to rotation or agitation for 15 seconds using a shakingplatform. The pattern of sample acquisition followed a row by rowsequence, which acquires samples sequentially from columns 1-12 of rowA, followed by acquiring samples sequentially from columns 1-12 of rowB, followed by acquiring samples sequentially from columns 1-12 of rowC, followed by acquiring samples sequentially from columns 1-12 of rowD, followed by acquiring samples sequentially from columns 1-12 of rowE, followed by acquiring samples sequentially from columns 1-12 of rowF, followed by acquiring samples sequentially from columns 1-12 of G,and finally followed by acquiring samples sequentially from columns 1-12of row H.

The total counts for each well were recorded and are reproduced in FIG.4. In addition, samples across each row and down each column of themulti-plate were analyzed to determine the average per well count(Avg.), the standard deviation (St. Dev.) and coefficient of variation(CV). In addition the well average from the entire plate was alsodetermined.

As shown in FIG. 4, the results show that far fewer particles weredetected from samples collected from center wells compared to thosearound the edges. For example, wells D7, E6, E7, F5-7, and G4-8 producedzero counts, whereas columns 1, 2 and 10-12 each averaged over 1000counts. The absence of counts is likely to have occurred by particlessettling to the bottom of the wells even after rotating or agitating themulti-well plate. The average count across the entire plate was 891.

Example II Improved Sample Consistency Found when Acquiring SamplesUsing a Spiral-Square Approach

The following example demonstrates improved consistency in particlecounts across the multi-well plate when acquiring samples using aspiral-square approach.

A suspension of silica beads was vortexed to ensure the beads wereequally distributed throughout the solution. An equal volume of thesuspension was aliquoted into each well of a 96 well plate to ensure anequal load across all wells. The NOVOCYTE flow cytometer (ACEABIOSCIENCES, San Diego, Calif.) was programmed to sequentially acquire10 uL of sample from each well and count the silica beads in eachacquired sample. Between acquiring samples from each well, the plate wassubjected to rotation or agitation for 15 seconds. The pattern of sampleacquisition followed a spiral-square configuration using the followingorder. D6, E6, E7, D7, C7, C6, C5, D5, E5, F5, F6, F7, F8, E8, D8, C8,B8, B7, B6, B5, B4, C4, D4, E4, F4, G4, G5, G6, G7, G8, G9, F9, E9, D9,C9, B9, A9, A8, A7, A6, A5, A4, A3, B3, C3, D3, E3, F3, G3, H3, H4, H5,H6, H7, H8, H9, H10, G10, F10, E10, D10, C10, B10, A10, A2, A1, B1, B2,C2, C1, D1, D2, E2, E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11, F11,F12, E12, E11, D11, D12, C12, C11, B11, B12, A12, A11.

The total counts for each well were recorded and are reproduced in FIG.5. In addition, samples across each row and down each column of themulti-plate were analyzed to determine the average per well count(Avg.), the standard deviation (St. Dev.) and coefficient of variation(CV). In addition the well average from the entire plate was alsodetermined.

As shown in FIG. 5, the results show more consistent particle countsacross the entire plate compared to the row-by-row approach of Example Iand no well had zero counts. Further, the average particle count acrossthe plat was 2672, which is a significant improvement of the resultsfrom Example 1.

Example III Improved Sample Consistency Found when Acquiring SamplesUsing a Nearest Well to Center Approach

The following example demonstrates improved consistency in particlecounts across the multi-well plate when acquiring samples using anearest well to center approach.

A suspension of silica beads was vortexed to ensure the beads wereequally distributed throughout the solution. An equal volume of thesuspension was aliquoted into each well of a 96 well plate to ensure anequal load across all wells. The NOVOCYTE flow cytometer (ACEABIOSCIENCES, San Diego, Calif.) was programmed to sequentially acquire10 uL of sample from each well and count the silica beads in eachacquired sample. Between acquiring samples from each well, the plate wassubjected to rotation or agitation for 15 seconds. The pattern of sampleacquisition followed a nearest well to center configuration using thefollowing order. D6, E6, E7, D7, C7, C6, D5, E5, F6, F7, E8, D8, C8, C5,F5, F8, E9, D9, B7, B6, D4, E4, G6, G7, G8, F9, C9, B8, B5, C4, F4, G5,H6, H7, E10, D10, A7, A6, D3, E3, G4, G9, B9, B4, C3, F3, H5, H8, F10,C10, A8, A5, A4, B3, G3, H4, H9, G10, B10, A9, D2, E2, E11, D11, C11,F11, F2, C2, A3, H3, H10, A10, B11, G11, G2, B2, D1, E1, E12, D12, C12,F12, F1, C1, A2, H2, H11, A11, B12, G12, G1, B1, A1, H1, H12, A12.

The total counts for each well were recorded and are reproduced in FIG.6. In addition, samples across each row and down each column of themulti-plate were analyzed to determine the average per well count(Avg.), the standard deviation (St. Dev.) and coefficient of variation(CV). In addition the well average from the entire plate was alsodetermined.

As shown in FIG. 6, the results show more consistent particle countsacross the entire plate compared to the row-by-row approach of Example 1and no well had zero counts. Further, the average particle count acrossthe plat was 2462, which is a significant improvement of the resultsfrom Example 1.

Example IV Improved Sample Consistency Found when Acquiring SamplesUnder Reduced Loads Using a Spiral-Square Approach

Since the above examples demonstrated improved consistency across theentire plate compared to the row-by-row approach, the spiral-squareapproach was also tested with reduced loads.

A suspension 5 μm polystyrene beads (Spherotech Accucount Beads) wasvortexed to ensure the beads were equally distributed throughout thesolution. An equal volume of the suspension was aliquoted into each wellof a 96 well plate to ensure an equal load across all wells. TheNOVOCYTE flow cytometer (ACEA BIOSCIENCES, San Diego, Calif.) wasprogrammed to sequentially acquire 10 uL of sample from each well andcount the silica beads in each acquired sample. Between acquiringsamples from each well, the plate was subjected to rotation or agitationfor 15 seconds. The pattern of sample acquisition followed aspiral-square configuration using the following order. D6, E6, E7, D7,C7, C6, C5, D5, E5, F5, F6, F7, F8, E8, D8, C8, B8, B7, B6, B5, B4, C4,D4, E4, F4, G4, G5, G6, G7, G8, G9, F9, E9, D9, C9, B9, A9, A8, A7, A6,A5, A4, A3, B3, C3, D3, E3, F3, G3, H3, H4, H5, H6, H7, H8, H9, H10,G10, F10, E10, D10, C10, B10, A10, A2, A1, B1, B2, C2, C1, D1, D2, E2,E1, F1, F2, G2, G1, H1, H2, H11, H12, G12, G11, F11, F12, E12, E11, D11,D12, C12, C11, B11, B12, A12, A11.

The total counts for each well were recorded and are reproduced in FIG.7. In addition, samples across each row and down each column of themulti-plate were analyzed to determine the average per well count(Avg.), the standard deviation (St. Dev.) and coefficient of variation(CV). In addition the well average from the entire plate was alsodetermined.

As shown in FIG. 7, the results show more consistent particle countsacross the entire plate compared to the row-by-row approach ofExample 1. A sampling error occurred in A5, which resulted in zerocounts. Variation between columns and rows was found to be at most 5%,which is much lower than the variation found in Example 1.

The detailed description set-forth above is provided to aid thoseskilled in the art in practicing the present invention. However, theinvention described and claimed herein is not to be limited in scope bythe specific embodiments herein disclosed because these embodiments areintended as illustration of exemplary improvements of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description which do not depart from thespirit or scope of the invention. Such modifications are also intendedto fall within the scope of the appended claims.

What is claimed is:
 1. A method of collecting cells from individualwells of a multi-well plate for flow cytometry, the method comprising:determining particle counts from each well of the multi-well plate byflow cytometry; ordering the wells from lowest particle count to highestparticle count; generating a sequential order for collection from theordering of wells to establish a collection pattern; and collectingparticles according to the collection pattern in a second multi-wellplate by flow cytometry.
 2. The method of claim 1, wherein the particlecounts are a number of counted cells and the collected particles arecollected cells.
 3. The method according to claim 1, wherein themulti-well plate is a 96 well plate, characterized as having rows A-Hand columns 1-12, further wherein the middle region is characterized asa well selected from the group consisting of wells D6, D7, E6 and E7. 4.The method claim 1, wherein the step of ordering the wells comprisesgrouping the wells by count ranges.
 5. The method claim 4, wherein thesequential order is such that groups of a lower count range are orderedbefore groups of a higher count range.
 6. The method of claim 5, whereinthe sequential order of wells within each group follow a same pattern.7. The method according to claim 1, wherein the step of generating thesequential order comprises establishing a successive square pattern or aspiral square pattern starting from the well having the lowest particlecount or from a well within a group of wells having a lowest countrange.
 8. The method according to claim 1, wherein the collectionpattern exactly follows the ordering of wells from lowest particle countto highest particle count.
 9. The method according to claim 1, whereinthe collection pattern deviates from the ordering of wells less than15%.
 10. The method according to claim 1, wherein the method furtherincludes rotating or agitating the multi-well plate between steps ofcollecting particles from different wells.
 11. The method according toclaim 1, wherein the method further comprises a second collectionpattern performed after the collection pattern.
 12. The method accordingto claim 1, wherein the second collection pattern is a meanderingpattern between two columns of wells.
 13. The method according to claim1, further comprising labeling the cells with labelled binding reagentsagainst one or more cell biomarkers.
 14. The method according to claim13, wherein the labelled binding reagents are fluorescently labelledantibodies or antibody fragments.